ES2975982T3 - Optical microscopy - Google Patents

Abstract

A method for enhancing interference contrast in interferometric scattering optical microscopy. The method comprises providing a particle detection region comprising a chamber or channel having a boundary defined by one or more interfaces, illuminating a particle in the particle detection region with coherent light using an objective lens such that the light is reflected from the interface and scattered by the particle, capturing the reflected light and scattered light using the objective lens, and providing the captured reflected and scattered light to an imaging device for visualizing interference between the reflected light and scattered light. The particle is illuminated with coherent light at an oblique angle to the interface. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Optical microscopy

Field

This invention relates generally to interferometric scattering optical microscopy.

The work leading to this invention has received funding from the European Research Council under the European Union's Seventh Frame Programme (FP7/2007–2013)/ERC grant agreement No. 337969. The project giving rise to this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 766972. The project giving rise to this application has received funding from the European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No. 674979.

Background

Interferometric scattering optical microscopy (iSCAT) is a technique in which an imaged particle is illuminated with coherent light and the signal results from interference between the light scattered by the particle and a reference light reflected from a nearby interface. This interference can result in signals that are amplified compared to other approaches, and the technique is capable of detecting single, unlabeled molecules such as biological molecules, e.g. proteins/protein complexes. However, the interference signal can be very small and difficult to detect, and typically the iSCAT setup creates spurious reflection which limits the achievable signal-to-noise ratio.

In more detail, the iSCAT signal can be separated into three components: the reflected laser intensity, the scattered intensity, and the interference between the two. The reflected laser intensity is dominant but does not convey any information, the scattered intensity is too weak to be detected, and only the interference is useful.

As described in Arroyo et al, “Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging,” Nature Protocols, vol. 11, no. 4, p. 617, 2016, “Alignment of the illumination beam is critical to the final image quality. Therefore, to ensure proper alignment, it is imperative that the [illumination] beam travels straight through the objective along its optical axis.” However, this arrangement raises the problem that despite using very high-quality antireflection coatings, a small fraction of the incident light is reflected from the back of the objective lens. Since the illumination laser has a short coherence length, on the order of hundreds of microns for a laser diode, this spurious reflection can reduce the signal-to-noise ratio and contrast by an order of magnitude. However, the use of a "long coherence length leads to interference from almost any reflection within the experimental setup, resulting in additional image noise" (ibid).

One way to increase contrast is described in Cole et al., "Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy" ACS Photonics 20174(2), 211-216, and also in WO2018/0l.

1591. It uses a partially transmissive phase mask that reduces the intensity of the background light reaching the detector, thus increasing the interferometric contrast. A very similar technique involving a partially transmissive mask is described in Liebel et al., "UltraSENSITIVE Label-Free Nanosensing and High-Speed Tracking of Single Proteins", Nano Letters 2017, 17(2), 1277-1281.

However, these techniques have drawbacks, for example, fringe artifacts and image distortion; they may also enhance the relative background of interface surface roughness. Counterintuitively, using a phase mask as described by Cole et al. improves contrast, but does not change the signal-to-noise ratio (this is because the signal is interferometric). Therefore, while the technique may help if the microscope is limited by the camera frame rate and can facilitate focusing, it does not improve the signal-to-noise ratio, which is the crucial parameter.

Further prior art background can be found in Goldfain et al. “Dynamic measurements of the position, orientation, and DNA content of individual unlabeled bacteriophages”, Journal of Phys. Chem. B, vol. 120(26), pp. 6130-6138, 2016; W02017/041809; W02015/059682; W02018/189187; W02018/047239; US2012/218629; and Konopka et al. “Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex” Plant Journal, vol. 53(1), pp. 186-196, 2008. EP3276389Al proposes a common path interferometric scattering imaging system. Joanna Andrecka et al: “Direct Observation and Control of Supported Lipid Bilayer Formation with Interferometric Scattering Microscopy,” ACS Nano, vol. 7 no. 12, 23 December 2013 (2013-12-23, pages 10662-10670), proposes interferometric scattering microscopy. WO 02/086578A2 proposes a dark-field microscope. Pache C et al: "Dark-field Optical Coherence Microscopy", Proceedings of s P iE/IS&T, vol. 7554, 25 January 2010 (201001-25), pages 755425-1 proposes dark-field optical coherence microscopy Matthias D. Koch et al: "Label-free imaging and bending analysis of Microtubules by ROCS Microscopy and Optical Trapping", Biophysical Journal, vol. 114, No. 1, 1 January 2018 (2018-01-01), pages 168-177 proposes ROCS microscopy in dark field mode.

Summary

Thus, in one aspect there is disclosed a method for increasing signal (interference) contrast in interferometric scattering optical microscopy. The method comprises providing a particle detection region having a boundary defined by one or more interfaces. The particle detection region may comprise a chamber or channel, or the particle detection region may comprise a liquid droplet on an optically transmissive surface, such as a coverslip. The method further comprises illuminating a particle in the particle detection region with coherent light using an objective lens such that the light is reflected from the interface and scattered by the particle. The method further comprises capturing the reflected light and the scattered light using the objective lens. The method further comprises providing the captured reflected and scattered light to an imaging device for visualizing interference between the reflected light and the scattered light. The objective lens has an optical axis. The imaging device may comprise imaging optics and, i.e., followed in an optical path, a detector.

In implementations, the method further comprises providing the coherent light at an oblique angle relative to the interface, to illuminate the particle. Thus, the method further comprises providing the coherent light to the objective lens off-axis, i.e., offset from the optical axis of the objective lens. The coherent light, for example, may be laser light or light from a narrowband LED source provided to the objective lens in a direction parallel to and offset from the optical axis.

Thus, in implementations, rather than being normal to the interface, the light illuminating the particle comprises a beam collimated at an oblique angle to the interface. Stated another way, an angle of incidence between the beam and a normal to the interface is greater than 0 degrees and less than 90 degrees; if the beam is not precisely collimated, the mean angle of incidence may be greater than zero degrees. In implementations, the angle of incidence is less than a critical angle at the interface.

Thus, in implementations, the coherent light provided at an oblique angle to the interface comprises a parallel or collimated beam. The beam may be collimated by the objective lens; thus, the coherent light may be focused on a back focal plane of the objective lens. The coherent light may propagate along an axis parallel to the optical axis of the objective lens, for example, the laser light may be aligned parallel to but offset from the optical axis of the objective lens. Conversely, the reflected and scattered light is captured in a direction along the optical axis of the objective lens.

In some implementations, an angle of incidence of the coherent light at the interface may be greater than an angle value at which, at the imaging device, an intensity of the reflected light is greater than an intensity of the light reflected from a rear surface of the objective lens. The angle of incidence may be less than this angle value plus 5, 10, or 20 degrees. For example, the angle of incidence is in the range of 1-30, 3-20, or 5-15 degrees, e.g., about 10 degrees.

It is not essential that the coherent light be focused precisely on a back focal plane of the objective, and the technique will work when the light illuminating the particle is not precisely collimated.

These techniques can greatly improve the signal-to-noise ratio and also the image contrast of the interference pattern. In implementations, the spurious reflection from the back of the objective lens can be substantially eliminated, collecting only the interfering reflection. While the laser is focused on the back focal plane, it is off-axis; the distance from the laser axis to the optical axis of the objective lens changes the angle of incidence of the light from the objective. Therefore, the spurious reflection from the back of the objective is not reflected directly into the camera. This prevents the spurious reflection from reaching the camera and does not reduce the signal-to-noise ratio; instead, mostly or only the interfering reflection, for example from a glass-water interface of the microscope, is reflected. Generally speaking, by illuminating at an oblique angle to the scattered light, the level of the reflected signal from the background can be reduced, increasing the signal-to-noise ratio as well as the interference contrast. The intensity of the light reflected from the interface is angle-dependent (as expected from Snell's law), but the signal-to-noise ratio is not. However, in the case of normal incidence, there is an additional contribution from spurious reflection which substantially reduces the signal to noise.

In some implementations, a rear surface of the objective lens is curved, e.g. domed, rather than flat. In this case, an additional benefit can be gained as the spurious reflection is at a slight angle to the incoming light. This deflects the spurious reflection, e.g., away from the rear optical/imaging device, e.g., onto the detector walls.

Some implementations of this technique are particularly, but not exclusively, suitable for imaging particles in solution, e.g., aqueous solution. Particles may comprise molecules or molecular complexes, such as biological molecules/complexes, e.g., proteins, protein complexes, or antibodies. More generally, particles may have a maximum dimension that is less than a wavelength of coherent light, or less than half this wavelength. Such particles may include metallic or other nanoparticles, colloid particles, polymeric particles, viruses, exosomes and other extracellular vesicles, proteins, and bioparticles in general.

As described below, in implementations the particles may be illuminated at the oblique angle using light polarized near the Brewster angle, through the objective lens, to collect interfering light, thereby reducing the level of background reflected signal and increasing interference contrast. While illuminating at an oblique angle may reduce the level of scattered light captured by the objective lens in the on-axis direction, contrast can generally be improved.

In case of very large oblique angles (with respect to the optical axis), the signal to noise ratio may decrease again. Therefore, the method may comprise adjusting the oblique angle to maximize the signal to noise ratio of the interference between the reflected light and the scattered light, i.e. to maximize the interference contrast. The interference contrast may be determined at the output of the imaging device, i.e. from the captured image of the interference. This is because the response of the imaging device, such as its inherent sensitivity/noise level, may also affect this trade-off. The imaging device may be a 1D or 2D imaging device, for example a camera. The optimal angle may also depend on the surface roughness of the interface.

As the angle of incidence increases away from 0 degrees, i.e., as the illumination becomes more oblique, the effect of spurious reflection from the back of the objective lens decreases, but the interference pattern becomes more distorted. Therefore, in some implementations, the oblique angle is selected such that the intensity of light from spurious reflections is equal to or less than the intensity of light reflected from the camera or channel interface (as measured at the imaging device), but not substantially greater than this value, for example, no more than 10, 20, or 30 degrees greater than this value.

One way to improve interference contrast without the need for such an oblique angle, and thus with less reduction in backscattered, is to polarize the illuminating coherent light. More particularly, in some implementations the method further comprises linearly polarizing the coherent light such that its electric field vector is partially or completely polarized with respect to a plane of incidence of the coherent light at the interface.

The illumination angle can then be further tuned to be close to the Brewster angle for the interface at the illumination wavelength, although it may not be exactly at the Brewster angle since some reflected light is needed for interference (although since the laser is typically not perfectly collimated there is still some reflection even if it is exactly at the Brewster angle). For example, the illumination angle can be substantially the Brewster angle, i.e. it can be close enough to the Brewster angle that the reflectance of the interface to coherent light is less than 10%, 1%, or 0.1%, or so that the intensity of the reflected light is greater than the scattered light.

A spatial filter or mask may also or instead be used to increase the contrast of the interference pattern, in particular by changing the intensity of the reflected light. The spatial filter or mask may be located in a focal plane of the microscope, more particularly in a focal plane of the reflected and scattered light captured, for example, in a back focal plane or Fourier plane of the objective lens.

In implementations, the reflected light travels from the optical axis of the objective lens in a direction opposite to the offset (from the optical axis) of the coherent light's optical path to the objective lens. Thus, the spatial filter may be off-axis, e.g., configured to selectively mask a region of the Fourier plane offset from the optical axis, and may be asymmetric about the optical axis leaving the illuminating coherent light and/or a region on the back focal plane axis unmasked. Typically, the center of the Fourier plane on/around the optical axis contains important information related to the desired interference signal, and implementations of the method facilitate spatial filtering away from this location into a less important portion of the Fourier plane. In general, the mask may have a low but non-zero transmittance.

In implementations, the spatial mask reduces the intensity of both the reflected light and the noise, and can therefore leave the signal-to-noise ratio relatively unchanged (see below for further discussion). However, reducing the intensity of the reflected light can increase contrast: because the signal is interferometric, the signal-to-noise ratio and contrast are somewhat decoupled from each other.

The iSCAT technique is based on interference between reflected and scattered light, and these are superimposed at the detector (imaging device), where an interference pattern is formed. At the back focal plane of the objective, the scattered light propagates along the back focal plane, but the reflected light is focused at a specific location (although it propagates through the detector because there is a Fourier transform between the back focal plane and the detector). At this specific location in the back focal plane of the objective, a mask can selectively attenuate the reflected light.

Therefore, in some implementations the mask (spatial filter) may be small, for example corresponding to a laser focal spot size (e.g. full width at half maximum), to reduce information loss. The mask may be disk-shaped. The mask may be partially transmissive, as described below, for example blocking more than 50%, 75%, 90%, or 94% of the light incident on the mask, but less than 100%, 99%, or 95% of the light, for example blocking about 99% of the light.

In some implementations, the mask may be provided on a substantially transparent support. If the back focal plane is not accessible, the mask may still be located in a focusing plane of the laser, which may be slightly offset from the back focal plane, i.e. the laser may be slightly out of focus (or the laser may be focused on the back focal plane, but the filter is slightly offset and with a slightly larger filtering region than it would otherwise have).

When used to image a particle in solution (rather than confined to a glass surface), the method may further comprise limiting the motion of the particle in a direction along the optical axis,

that is, the z direction, less than a distance 2^ÁTÁ’^2'o^4’ where A where is the wavelength of the coherent light (in the solution). This can be done, for example, by appropriately sizing the chamber or channel, e.g., A

providing a pair of opposite boundaries spaced by this distance in the z direction. A range of <2> represents a range from maximum contrast to zero contrast in the interference image. In this way restricting motion A

of the particles, for example, towards inside 4', facilitates tracking of the particle because the particle is inhibited from disappearing from view (and reappearing elsewhere). However, it is not essential that the motion in the z direction A A

be limited to A or 4, to obtain any benefit. As described above, the method uses scattering and not, for example, fluorescence. Therefore, the particle can be a non-fluorescent particle. The particle can be label-free. In general, the method is used off-resonance, i.e. far from an absorption peak/edge of the particle. However, this is not essential and in principle light close to an absorption peak/edge can be used.

In a related aspect, an interferometric scattering optical microscope is described. In implementations, the microscope comprises a particle detection region, for example, comprising a chamber or channel or a droplet on a surface, the particle detection region having a boundary defined by an interface, a coherent light source such as a laser, and an objective lens for directing the coherent light to illuminate a particle in the particle detection region such that the light is reflected from the interface and scattered by the particle. The objective lens has an optical axis and is configured to capture the reflected light and the scattered light. The microscope further comprises an imaging device configured to visualize the interference between the reflected light and the scattered light. The coherent light illuminating the particle is at an oblique angle relative to the interface. Thus, an optical path of the coherent light to the objective lens is offset from the optical axis of the objective lens. In implementations, the coherent light may be provided to the objective lens in a direction parallel to and offset from the optical axis.

In some implementations, the coherent light source is linearly polarized as described above.

Thus, the coherent light illuminating the particle may be partially or completely p-polarized with respect to a plane of incidence of the coherent light on the interface.

In some implementations, the coherent light source is linearly or circularly polarized, and an analyzer with an orthogonal polarization is provided in an optical path between the particle and the imaging device. This may provide a signal that depends on a shape asymmetry of the particle, but with a reduced signal-to-noise ratio.

An optical path between the objective lens and the coherent light source may include a beam splitter to separate the returned reflected and scattered light from the incident illumination light, but this is not essential since in implementations these two light paths are at different spatial positions relative to the optical axis. The imaging device may comprise a 1D or 2D image sensor, for example an EMCCD (electron-multiplying charge-coupled device) sensor or a fast CMOS camera, and an optical element such as a lens or mirror to focus the returned light onto the sensor.

In some implementations, the microscope/method is configured to process the image interference to determine a difference between the image interference at two different times, e.g., using a processor under stored program control. For example, a difference frame may be determined between two interference image frames, or groups of frames, captured by the imaging device. The difference frame may be Fourier transformed, e.g., by the processor, to determine a Fourier image. This may then be processed to characterize the particle or a solution of the particles, e.g., by selecting a portion of the Fourier image within an expected scattering circle. The expected scattering circle may comprise a region of the Fourier image within which the interference between reflected light and scattered light from the particle(s) is/should be confined. The region of the Fourier image within the expected scattering circle can be processed, for example, to determine a level or measure of scattering in this region, to characterize the particle(s) or a solution of the particles.

Other features of the microscope may correspond to those of the method described above, i.e. the microscope may include systems/devices to implement these features.

Drawings

These and other aspects of the system will now be described in greater detail by way of example only, with reference to the accompanying figures, in which:

Figure 1 shows a schematic diagram of an interferometric scattering optical microscope (iSCAT).

Figures 2a and 2b illustrate, respectively, normal illumination and oblique angle illumination techniques for the iSCAT optical microscope of Figure 1, and Figure 2c shows a modification of the microscope of Figure 1 to implement oblique angle illumination in accordance with one embodiment of the invention.

Figures 3a and 3b illustrate, respectively, images captured using the normal and oblique incidence illumination techniques of Figure 2.

Figure 4 shows, schematically, details of the optical geometry of another example of an iSCAT microscope with oblique angle illumination.

Figure 5 shows a graph of contrast and intensity of the iSCAT signal seen by the camera versus incidence angle.

Figure 6 shows example interference images of a strongly scattered particle (top row) and the corresponding predicted interference patterns (bottom row).

Figure 7 shows Fourier transforms of interference images captured at different incidence angles on an iSCAT microscope.

Figure 8 shows the optical geometry related to hologram generation.

In the figures, similar elements are indicated by similar reference numbers.

Description

Referring to Figure 1, this shows a schematic diagram of an interferometric scattering (iSCAT) optical microscope 100. The microscope comprises a continuous wave laser 102 that provides a coherent light beam 104 to a focusing element, lens 108, which focuses the beam onto the back focal plane 110 of a microscope objective lens 112. In this example, the laser is in the visible, at 445 nm, and its optical path is bent by the mirror 106 toward the lens 108. The laser may have a coherence length greater than 50 pm or 100 pm and/or less than 100 mm or 10 mm.

The objective lens 112, which may be an oil immersion objective, may be adjusted to provide generally uniform illumination in a detection region 114. In the example of Figure 1, the detection region 114 is defined by a chamber or channel 116 with upper and lower surfaces 116a,b, made of, for example, glass or polymer. The chamber/channel 116 may contain a solution 118, such as an aqueous solution (i.e., water), containing one or more particles 120 to be imaged. In some other arrangements, the particles to be imaged may be immobilized on a glass surface, such as the surface of a coverslip. In some implementations, the distance between the surfaces 116a,b may be on the order of one-half the wavelength of coherent light in the solution, to maintain a view of the imaged particle m.

The illumination is reflected from a reflective interface 122, for example, between the bottom surface 116a of the chamber/channel 116 and the solution 118. The illumination is also scattered by the particle(s) 120. Both the reflected light and the scattered light are captured by the objective lens 112, returned along the illumination optical path, and directed to a separate path 124 by a beam splitter 126. Images of the reflected light and the scattered light are then obtained. For example, the reflected light and the scattered light are focused onto an image sensor 130 by imaging optics such as a lens 128. The image sensor (camera) may be, for example, a CMOS image sensor or an EMCCD image sensor; it may have a frame rate sufficient to capture and track the motion of an imaged particle.

Figure 2a shows details of a normal incidence illumination technique for the iSCAT optical microscope of Figure 1. The coherent light beam 104 may be unpolarized; that is, it may comprise light of two orthogonal linear polarizations 202a,b or, more generally, it may be elliptically polarized. In the particular example illustrated, the solution flows along channel 116, which has a height (z direction) of about 450 nm and a width of about 5 pm.

Figure 2b shows details of an oblique angle illumination technique for the iSCAT optical microscope of Figure 1. An optical axis of the objective lens 112 lies along the direction of line 200, which represents the reflected light and scattered light captured by the objective. The coherent light beam 104 illuminating the detection region 114 is off-axis, i.e., shifted away from the optical axis 200; one axis of the beam 104 may be parallel to the optical axis 200. Thus, the beam 104 defines an angle 0i with respect to the optical axis 200 at the exit point of the objective, which forms an oblique angle with respect to the reflective interface 122. (The direction of this beam is subsequently modified by refraction, but the illumination nevertheless remains at an oblique angle with respect to the interface 122.)

By illuminating the imaged particle(s) at an oblique angle, the signal level, i.e. the interference between scattered and reflected light, increases compared to the background level of reflected light. The proportion of these components can be adjusted by adjusting the oblique angle, if the angle 0i is too large, the intensity of scattered light along the direction is reduced; if it is too small, the reflected light reduces the signal to noise ratio. The optimal angle can be found by experiment; in general, it may depend on the background noise of the camera.

In some implementations, the oblique illumination may be partially or completely linearly polarized, in particular p-polarized, to reduce the level of reflected light from interface 122. At the Brewster angle, the level of reflected light from interface 122 is substantially reduced to zero (actually greatly reduced but not to zero because the laser is not perfectly collimated). In practice, the oblique angle may be slightly offset from the Brewster angle to provide some reflected light to generate the interference. Controlling the polarization of the illumination is advantageous as it allows the reduction in intensity of the reflected light to be somewhat decoupled from the reduction in intensity of the scattered light captured along direction 200.

Figure 2c shows an example implementation of the oblique angle illumination technique of Figure 2b in the context of the microscope of Figure 1.

Thus, Figure 2c shows a schematic diagram of an iSCAT 200 optical microscope that includes a laser 202 for generating a coherent light beam 204. The beam 204 is focused, for example, by a lens 206, e.g., onto a back focal plane of the objective lens 112. A reflector, diagonal mirror 210, directs the coherent light beam 204 off-axis toward the objective lens 112, i.e., the beam 204 is displaced away from an optical axis of the objective lens 112, direction 200 in Figure 2b. The off-axis beam 204 is transformed by the objective lens 112 into a collimated beam 204a that illuminates the reflective interface 122 at an oblique angle.

Figures 3a and 3b illustrate, respectively, images captured using the normal and oblique incidence illumination techniques of Figure 2. It can be seen that Figure 3b, captured using oblique illumination, shows a much higher interference contrast than Figure 3a and, consequently, much more image detail. In practice, to detect a particle as a single molecule, differential detection is used, for example, the background subtraction technique can be employed.

As described in more detail below, a captured interference image of a particle can be called a hologram since what is captured is an interference pattern. However, the hologram usually has a bright (or dark) central region (depending on the phase), which gives the appearance of a particle.

In implementations, interference contrast may refer to, for example, a ratio of light intensities between bright and dark regions of an interference pattern (from the iSCAT microscope), or a ratio of image brightness values between bright and dark regions of an interference image (from the iSCAT microscope).

More details of some example implementations are described below.

Example implementations

In interferometric scattering (iSCAT) implementations, a high-intensity receiver wave interfered with a scattered signal, allowing the system to operate in a shot noise-limited regime. The dependence of the signal on the particle diameter is expressed in the third power; a scattering tag, typically a gold nanoparticle, can be used to increase the scattering cross section. iSCAT can be used to detect particles in solution, for example in water, allowing their diffusion coefficient to be estimated. However, in this application the integration time is limited as the particle must not diffuse beyond the width of the point spread function, although subcritical incidence angles can decrease background scattering and improve the signal-to-noise ratio.

Interferometric scattering (iSCAT) works by collecting both scattered light and reflected light at the detector. Since they both have the same wavelength, they will interfere. The collected intensity (Pcol) is the result of the reflected electric field (Er) and the scattered electric field (Es):

where the incident power is Pinc and the reflected power is Pref = Pinc |r|2 with r the reflection coefficient of the electric field. The interference power is P¡nt = Pinc2|r| |s| cos O where s is the scattering coefficient and O is the phase difference. The reflection coefficient (r) is given by the Fresnel equations (see below) and the scattering cross section a= |s|2 is given by Rayleigh Scattering (see below). Usually |s| « |r|, so the final |s|2 term is negligible. The reflected term Pref can be eliminated if it changes more slowly than the interfering term P¡nt. This is the case for a diffusing sample or for junction events, for example. The contrast depends on the reflectivity:

But surprisingly, the signal-to-noise ratio is not: assuming the system is noise-limitedPruidoKjPcot ~7pref■ The noise is given by the signal-to-noise ratio (SNR) is:

which is independent of the reflected intensity. The dispersion coefficient can be improved by decreasing the laser wavelength; otherwise, it seems that the only way to improve the signal-to-noise ratio is to increase the laser power or use longer integration times, although longer integration times are limited by drift.

Oblique lighting

Figure 4 shows, schematically, details of the optical geometry of an additional example of an iSCAT 400 microscope as described herein.

As before, a beam 204 of coherent light, for example, from a laser 202, is reflected by a semi-transparent mirror or beam splitter 410, in this example offset away from the back focal plane 110 of the objective lens 112. The beam 204 is focused at a back focal plane of the objective lens 112, collimated by the objective lens 112, and, in this example, passes through a refractive index matched oil 412 to the reflective interface 122. The beam 204 is offset from the optical axis 200, and exits the objective lens 112 as a collimated beam 204a at an oblique angle to the glass-water reflective interface 122.

In this example, the reflective interface 122 comprises a glass-water interface formed by the interface between a glass wall 414 of a chamber or channel and an aqueous solution in region 416. As the beam 204 traverses this path, small amounts of light are reflected off other interfaces in the path, such as interfaces in the objective lens 112; the index-matched oil 412, matched, for example, to a refractive index of the objective end element, reduces the number of interfaces that the illuminating light sees in the path. The intensity of the reflections from the glass oil is nearly zero because the oil is index-matched.

At the glass-water interface 122 the illuminating light is separated into transmitted and reflected components. The transmitted illumination reaches one or more particles 120 in the aqueous solution that scatter the illumination. The scattered light is scattered in many directions, for example, approximately about a sphere (although this depends on factors such as the shape and configuration of the scattering particle and the polarization of the light). Some of the scattered light is collected by the objective lens 112 and provides a scattered light beam 430 that in implementations extends across the back surface of the objective and travels in the direction 200 along the optical axis.

The reflected light returns along path 418, at an angle of reflection coinciding with the angle of incidence, to objective lens 112. Objective lens 112 focuses the reflected light at back focal plane 110 into a region offset away from optical axis 200, on the opposite side of the axis to the illumination beam 204. This reflected light interferes with scattered light at detector, e.g., camera 130, generating interference fringes.

In some implementations, a spatial filter 422 may be located at or near the back focal plane 110 of the objective lens 112 to further enhance contrast. For example, the spatial filter 422 may comprise a disk located at the focus of the reflected beam, with an extent corresponding to an extent of the focal point of the reflected beam or greater. The spatial filter may comprise a light attenuating mask 422 on a substantially transparent mask holder 420, e.g., a mask holder having the shape of a half disk (on the side of the optical axis that includes the reflected beam). A method for determining a degree of transmissivity of the light attenuating mask 422 is described below.

Spurious and Interferential Reflections

The above analysis assumes that reflection interferes with the scattered intensity. This is only true if the path difference is smaller than the laser coherence length (see below), typically hundreds of micrometers (for laser diodes). However, laser illumination is reflected from the back of the target and at every interface it crosses. Even targets with anti-reflection coatings do not have 100% transmittance, in which case the collected intensity can be rewritten with the reflected intensity separated into interfering and spurious reflection components:

where the spurious reflection electric field Ers does not interfere with the scattered signal or the reflected signal from the interface; the interfering reflection electric field is E„. Therefore, the above signal-to-noise ratio (SNR) is reduced if |rs|2 >> |n|2:

In the cases considered here only the reflection at the reflecting interface 122, for example a glass-water interface, will be an interference reflection. Therefore, Fresnel's law (see below) can be used to distinguish the two types of reflection.

Figure 5 shows a graph of the iSCAT signal contrast and intensity as seen by the camera versus the angle of incidence. The measured intensity (dots) comprises the reflected intensity, fitted with a line, and a component due to spurious reflections near the normal (0 degrees), where the dots diverge from the line. At angles greater than that at which the dots are located (in this example, around 10 degrees), the effect of spurious reflections essentially goes to zero (looking at the logarithmic scale). Two regions of the image are analysed, a region with a strong scatterer (positive control, upper crosses) and a region with no apparent scatterers (negative control, lower crosses). Without spurious reflections, the distance in logarithmic space between the scatter and the background should be constant and the value should depend on the reflected intensity (the signal, and therefore also the signal-to-noise ratio, is related to the difference between the upper and lower crosses). Below about 10 degrees, this is not the case and the signal-to-noise ratio goes to zero at small angles (i.e. the upper and lower crossings converge).

The line therefore represents the intensity fitted using the Fresnel equations. The points, which are the measured intensities, follow this line for high angles, but deviate from the fit at normal incidence due to spurious reflections. The effect can be seen in the contrast of a region with strongly scattered particles and a background region: both the noise and the scattered intensity are proportional to |r| and both contrasts follow the shape of the line, but not at normal incidence where the contrast is masked by the spurious reflection.

Image deformation

The image is formed by interference between scattered light and interfering reflection. The shape of the interference pattern can be calculated from the wavelength and angle of the laser (see below).

Figure 6 shows examples of interference images of a strongly scattered particle (top row) and the corresponding predicted interference patterns, i.e. holograms (bottom row) for different incidence angles of the illumination beam. At normal incidence, the strong spurious reflection hides the signal, but at higher incidence angles, the spurious reflection is not present but the image is progressively distorted as the circular pattern becomes more elliptical. To minimize distortion, the incidence angle can be chosen to be at or just above the angle where the spurious reflection manifests itself. This can be defined as an angle at which, at the imaging device, the intensity of the reflected light is greater than the intensity of the light reflected from a back surface of the objective lens. To minimize distortion, the angle cannot be greater than this angle plus, for example, 10 degrees.

Greater contrast

Decreasing the reflected intensity will increase the contrast, C, as shown in the above equation for C. Surprisingly, it does not change the signal to noise ratio, SNR, as shown in the above equation for SNR. Improving contrast is useful as it decreases the strain on the camera's dynamic range and can help the user glimpse the scattered signal while watching raw video, for example while focusing. When using oblique illumination, two techniques can be employed to achieve this. First, Brewster's angle of incidence can be used to decrease the reflected intensity, although this typically requires a fixed polarization and angle. Additionally, or instead, a spatial filter can be placed in the back focal plane, as shown in Figure 4. This is not possible at normal incidence as both the incident and reflected lasers pass through the same point, but at oblique incidence it is only possible to filter out the reflected intensity as the incident and reflected light are spatially separated.

Fourier plane

An objective lens creates a Fourier transform of the focal plane into the back focal plane. The Fourier plane can be recreated from the image by taking a Fourier transform of the interference image, i.e. a hologram, e.g. a fast Fourier transform (FFT) as described below.

Figure 7 shows fast Fourier transforms of interference images (holograms) captured at different incidence angles on an iSCAT microscope. The images are of a 100 nm particle (left and middle images) and a 40 nm particle (right images). Since phase information is lost when the light reaches the camera, the reconstructed Fourier plane shows a symmetrical pattern. The distance between the centers of the circles in the middle and right images is proportional to the translation of the illuminating laser from the optical axis (later AX).

In Figure 7, the dashed circle indicates the Abbe diffraction limit (imposed by the numerical aperture). The solid line indicates the expected dispersion circle, calculated as described below, here referred to as the Fourier circle. The cross indicates the position of the reflected laser. The size of the reflected laser spot (most easily seen in the lower right image) can be reduced by finely focusing the 128 lens so that the focal length matches the distance from the lens to the back focal plane of the objective; it can then be filtered out of the Fourier transform.

At normal incidence (left images), the reflected laser dominates both the single-frame and dissimilar particle images, while it is barely visible at higher angles: with increasing incidence angle (middle and right images), essentially only the interfering reflection is visible. In the particular example shown, the scattering signal from the 100 nm particle is stronger than the camera noise, while that from the 40 nm particle is buried in the noise.

Since the scattered signal is contained within the Fourier circle, the rest of the image (outside the expected scattering circle) can be filtered out, greatly reducing the shot noise. The residual smear from the laser reflection can be reduced if a spatial filter is used as described above.

In Figure 7, the top row shows the FFT of a single frame and the bottom row shows the FFT of a difference between two, for example, consecutive frames (a difference frame); in this example, the frame rate is set to 125 FPS by summing consecutive frames. Therefore, the bottom row shows only dynamic elements of the captured images, e.g. some parts capture interference images related to moving particles.

The signal inside the Fourier circle, for example the amplitude or intensity integrated over the area of the Fourier circle, in the FFT of a differential frame, can be used as a (one-dimensional) measure of the amount of scattering in a frame. This can be used as a measure of the concentration and/or size of the particles in a sample of the solution containing the particles; the measurement can be calibrated to determine a value for the concentration and/or size of the particles.

Example of device configuration

Referring again to Figure 4, in an exemplary implementation, a laser is reflected off a semi-transparent mirror and focused onto the back focal plane of a target. The laser or an optical element for guiding the laser may be mounted on a stage to allow a translation of the laser focal spot into the back focal plane while keeping the laser parallel to the optical axis of the target. An oil immersion target may be used to avoid creating interfaces between the target and glass (e.g., coverslip). A lens 128, e.g., a tube lens, focuses the image onto a camera 130.

The laser can be focused onto the back focal plane, so that it exits the target as a collimated beam. The location of the back focal plane can be found by minimizing the size of a spot on a distant screen (e.g. a wall or ceiling) while changing the position of the focal spot. The reflected intensity will then also be focused onto the back focal plane, and an optimal tube lens position can be found by imaging the back focal plane and minimizing the reflected spot. This can be done by using another tube lens with half the focal length to image the Fourier plane directly or by applying a fast Fourier transform to the recorded image.

The laser must be parallel to the optical axis of the objective. An offset between the optical axis and the laser axis can be applied to change the angle of incidence of the illumination. A graph of the type shown in Figure 5 can be drawn, for example, by recording the intensity of the reflected signal and fitting it to the Fresnel equation. In this way, the range of angles at which a spurious reflection is visible can be identified and an oblique illumination angle for the iSCAT microscope, i.e. an angle of incidence for the illumination beam, can be selected accordingly.

In some implementations, the angle of incidence (90 degrees relative to the oblique angle of illumination of the reflective interface) may be large enough to substantially eliminate spurious reflection but not significantly larger than this to reduce interference image distortion - for example, in the range 1-30, 3-20 or 5-15 degrees, e.g., about 10 degrees in the example in Figure 5.

A dynamic part of the image can be extracted by taking a difference between two interference image frames or between two sets of frames, e.g. groups of averaged frames. As described above, the Fourier circle can be used to remove a large part of the camera shot noise that lies outside the circle (in the example in Figure 7, the region inside the solid line is retained along with its symmetric counterpart). The noise created by the laser reflection can be removed since it is well localized in the Fourier plane. Note that if the tube lens is not positioned correctly so that the distance from the objective to the tube lens is equal to the focal length of the tube lens, the location of the reflection noise will be blurred in the Fourier plane.

The spatial mask, if used, should ideally be located in the back focal plane. The reflected laser should pass through a low transmittance region while the rest of the back focal plane should be as transparent as possible.

In general terms, the goal is to have, at the same time, a high signal-to-noise ratio (SNR) and a high contrast of the interference pattern. To increase the contrast, the reflected intensity should be reduced as much as possible. In principle, this does not affect the SNR if the noise is limited to the noise. However, as the intensity is reduced, eventually other sources of noise dominate and the SNR begins to decrease. Therefore, the transmittance of the mask should be reduced until other background noise begins to become apparent, for example, until the shot noise is at the same level or just above the background noise, for example, no more than 2x or 10x the background noise. Background noise comes from many sources, for example, stray illumination, electrical noise in the detector (camera), etc.

In the interference pattern, if the mask were transparent, the shot noise would dominate the noise component of the detected interference signal (provided the laser source is bright enough). Shot noise scales as n, where n is the number of photons. As the transmittance of the mask decreases there are fewer photons, implying a reduced level of shot noise. However, with very few photons the other sources of noise become apparent. It may be advantageous to reduce the transmissivity of the mask to block as much reflected light as possible while still allowing the shot noise to dominate. In practice this may be a transmissivity on the order of 10%, but this figure depends significantly on implementation details. As a practical technique, one can decrease the transmissivity of the mask until background noise is observed, and then decrease this value slightly.

If the back focal plane is not accessible, the focal position of the laser focus can be adjusted slightly so that the focus of the reflected beam is located on the spatial mask. Alternatively, the laser can be focused on the back focal plane, but the filter is shifted slightly away from it, thus having a slightly larger filter region than would otherwise exist.

The described techniques can improve iSCAT by removing spurious reflection that limits the signal-to-noise ratio. Contrast can also be improved in a number of ways, for example by using the Brewster angle and/or a spatial filter at or near the back focal plane.

To help understand the techniques, it is helpful to describe some of the underlying theories.

Dispersion

Rayleigh scattering describes the scattering of small particles, typically much smaller than the wavelength. The intensity Is at a distance X from a particle with a scattering angle 0 is proportional to the incident intensity Iq:

where nm is the refractive index of the surrounding medium, np is the refractive index of the particle, <y> is the wavelength of the coherent light, and r is the radius of the particle. Integration over the angles gives the scattering cross section a. Asymmetry can be detected by using a cross-polarization arrangement to detect changes in polarization caused by asymmetry of the particle shape; this signal is much weaker than the scattered intensity.

By focusing the laser onto the back focal plane of the objective, oblique illumination can be applied to the sample. If the particles of interest are relatively far from the surface, the technique can be operated at subcritical angles to obtain some intensity, i.e. a signal from an interior region of a channel or chamber or from the interior of a droplet on a coverslip. For an oil immersion objective, the objective focal length Fobj can be calculated from the tube lens focal length Fti and the magnification (Ma) using:

where the refractive index of the immersion oil is born. Then, the angle of incidence can be calculated from the distance of the laser position at the rear aperture (back focal plane) from the optical axis (AX):

s

where sine can be used instead of tangent because the target is corrected for Abbe's sine condition. The angle in the water can then be calculated from Snell's law:

The coherence length is the maximum distance over which light is coherent with itself and depends on the spectral width of the light source, e.g. laser. For SCAT, a short coherence length can help avoid interference from many reflections. Laser diodes have relatively broad spectra, e.g. AX=2 nm; the coherence length L is given by:

and for a typical laser diode it can be as short as 100 pm. Therefore, a reflection from a rear surface of the objective lens will not normally contribute to the interference signal.

The Fresnel equations describe the reflected and transmitted coefficient from medium 1 to medium 2 depending on the angle of incidence. They are, for s-polarized light:

And for p-polarized light:

At the Brewster angle there is no reflected light for p polarization, although light from a real laser is typically not perfectly collimated or polarized, so some reflection will still occur.

The objective lens transforms circular waves into collimated beams and vice versa. This is mathematically similar to performing a Fourier transform and the image in the back focal plane of an objective lens is the Fourier transform of the focal plane.

Figure 8 shows the optical geometry involved in hologram generation. The "Glass" region is assumed to have a reflection index equal to that of water, so that the relevant lengths can be compared; this does not apply if the focal plane is below the interface.

Referring to Figure 8, the interference image is created by the path difference between the reflected and scattered light. The optical system (e.g. lens 128) is assumed to transmit the magnified hologram to the camera. The scattered light travels a distance d0 from the glass-water interface to the particle, and a distance r from the particle to the focal plane. The reflected light does not cross the focal plane, but the reflection appears to come from the focal plane, at a distance Ar, which is negative for small angles of 9W, where 0w is the angle in water. Here the coherence length is assumed to be large enough for parallel rays to have the same phase. The path difference is A = As- Ar= d0 r - An The geometry gives, for the reflected path distance:

where Zf is the distance from the focal plane of the target to the reflecting interface and z is the distance from the particle to the focal plane. Then, for the scattered path difference:

Therefore, the difference in path length is:

and defining d as:

This can be rewritten as:

with

This defines an interference image or hologram that has an ellipse of eccentricity e = |sin 9w| centered around (x,y) = (dtan9w, 0). The innermost ellipse is centered around the smallest possible value of A, which corresponds to x= |z| tan 0w. This is where the light would arrive if it were reflected by the particle. From the Pcol equation, the normalized hologram is found to be:

Where is given by the Rayleigh scattering equation above. The intensity must be integrated over a pixel:

where the solid angle of the pixel is given by:

The spatial frequencies of the hologram are led by the term cosO, with In the case where z = 0, x, y, and r are linked by x = rcos0, y = rsen0.

The Fourier transform of:

It comprises two rings with radius and center ±sin0wnm/A. If z £0 the frequencies will be located inside the circle.

The numerical aperture (NA) is a property of the objective lens and describes the maximum angle of incidence 0max in a medium with refractive index n that is accepted by the objective. It is given by NA = nsin0max and is related to 2NA>

the maximum resolvable frequency by the Abbe diffraction limit This can be used to

calculate the useful pixel size (px) as the maximum frequency that can be resolved asl and therefore

Therefore, the pixel size must be less thanv r To detect the Fourier circle described above the numerical aperture must be such <f maxAbbey thereforeN

Applications

Applications of the described systems include locating and/or tracking one or more particles.

For example, by fitting a captured interference image to the above equation for an elliptical hologram, a particle's depth or 3D position can be determined from a particle's distance from the objective lens focal plane and/or from the reflective interface (z-up distance).

In another application, a particle can be tracked in one, two, or three dimensions, in which case the camera may be a video camera. This may allow a particle to be characterized by determining a diffusion coefficient for the particle, which depends on an effective or hydrodynamic size of the particle. The diffusion coefficient depends on the mean square displacement of a particle per unit time.

In some implementations, a particle may be confined in the z-direction, for example in a microfluidic channel. This can help particles to more frequently be found in or across the focal plane of the target, where the signal is highest.

Image processing can be used to enhance the signal, for example by averaging interference image frames to determine a background which is subtracted. Since the signal arises from interference, after such subtraction the signal can be positive or negative, which must be taken into account when averaging. For example, the signal can be averaged over a time shorter than the time it takes to travel a quarter wavelength in the z direction. When background subtraction is employed, a static, for example, bound particle can be detected by moving the channel/chamber in which the particle is bound.

In general, it is desirable to minimize vibrations, for example by using an anti-vibration table, as these move interference patterns and can eliminate the signal.

The chamber or channel boundary defining the interface is typically an internal channel/chamber boundary, i.e. an interface with the solution. In principle, however, it could be an external channel/chamber boundary. In principle, the interface could be curved, in which case coherent light would illuminate the particle at an oblique angle to a plane tangent to the interface at the illumination point. In principle, the illumination could be near or at an absorption edge of an imaged particle.

Many alternatives will occur to the skilled person. The invention is not limited to the embodiments described and encompasses modifications obvious to those skilled in the art which are within the scope of the appended claims.

Claims (15)

1. A method for increasing signal contrast in interferometric scattering optical microscopy, the method comprising: providing a particle detection region (114) having a boundary defined by an interface (122); illuminating a particle in the particle detection region (114) with coherent light using an objective lens (112) such that the light is reflected from the interface (122) and scattered by the particle; capturing the reflected light and the scattered light using the objective lens (112); and providing the captured reflected and scattered light to an imaging device (130) to obtain interference images between the reflected light and the scattered light; wherein the objective lens (112) has an optical axis, the method further comprises: providing coherent light to the objective lens (112) offset from the optical axis of the objective lens (112). so that a mean angle of incidence of the coherent light illuminating the particle forms an oblique angle with respect to the interface (122). 2. A method as claimed in claim 1, further comprising linearly polarizing the coherent light illuminating the particle such that the coherent light illuminating the particle is partially or completely p-polarized with respect to a plane of incidence of the coherent light on the interface (122). 3. A method as claimed in claim 1 or 2, wherein the oblique angle substantially defines the Brewster angle for coherent light at the interface (122). 4. A method as claimed in claim 1, 2 or 3, further comprising adjusting the oblique angle to maximize the signal to noise ratio of the interference between the reflected light and the scattered light. 5. A method as claimed in any one of claims 1-4 comprising providing the particle in solution (118) and using a chamber or channel (116) with a pair of opposing boundaries configured to restrict the- A . A. -motion of the particle in a direction along the optical axis to a distance less than 2 or 2, where A is the wavelength of coherent light. 6. A method as claimed in any one of claims 1 to 5, wherein the particle comprises a biological molecule in aqueous solution. 7. A method as claimed in any preceding claim, further comprising processing the reflected interference to determine a difference between the reflected interference at two different times, and Fourier transforming the difference to characterize the particle or a solution of the particles. 8. An interferometric scattering optical microscope, the microscope comprises: a particle detection region (114) having a boundary defined by an interface (122); a source (202) of coherent light; an objective lens (112) for directing the coherent light to illuminate a particle in the particle detection region (114) such that the light is reflected from the interface (122) and scattered by the particle; wherein the objective lens (112) has an optical axis and is configured to capture reflected light and scattered light; and an imaging device (130) configured to visualize the interference between the reflected light and the scattered light; and wherein an optical path of the coherent light to the objective lens (112) is offset from the optical axis of the objective lens (112) such that an average angle of incidence of the coherent light illuminating the particle forms an oblique angle with respect to the interface. 9. A microscope as claimed in claim 8, wherein the coherent light source is linearly polarized such that the coherent light illuminating the particle is partially or completely p-polarized with respect to a plane of incidence of the coherent light on the interface (122). 10. A method or microscope as claimed in any preceding claim, wherein the coherent light illuminating the particle at an oblique angle comprises a collimated beam of coherent light, and wherein the coherent light provided to the objective lens (112) is focused in a back focal plane (110) of the objective lens (112). 11. A method or microscope as claimed in any preceding claim, wherein an angle of incidence of the coherent light at the interface (122) is between i) an angle value at which, in the imaging device (130), an intensity of the reflected light is greater than an intensity of the light reflected from a rear surface of the objective lens (112), and ii) the angle value plus 10 degrees. 12. A method or microscope as claimed in any preceding claim, further comprising an off-axis spatial filter in or adjacent to a Fourier plane of the objective lens (112) configured to selectively mask a region of the Fourier plane displaced from the optical axis in a direction opposite to the displacement from the optical axis of the coherent light optical path to the objective lens (112). 13. A method or microscope as claimed in any preceding claim, wherein a spatial filter mask is positioned at or adjacent to a back focal plane (110) of the objective lens (112) or at a focal plane of the coherent light, and is configured to mask a region located at the focus of a reflection of the coherent light from the interface (122) and/or in which the coherent light is polarized, and an analyzer with an orthogonal polarization is provided in an optical path between the particle and the imaging device (130). 14. A microscope as claimed in any one of claims 8 to 13, further comprising a processor for processing the image interference to determine a difference between the image interference at two different times, and Fourier transforming the difference to characterize the particle or a solution of the particles. 15. A method or microscope as claimed in any preceding claim, wherein a rear surface of the objective lens (112) is curved and/or wherein the particle detection region comprises a chamber or channel (116).